This invention relates to methods for regenerating grape plants, and grape germplasm produced using such methods.
Grapevines are a deciduous temperate fruit crop of ancient origin. Grape production (65xc3x97106 metric tons) exceeds that of any other temperate fruit crop, and ranks third after Citrus and banana production. In addition, due to its uses for fresh fruit, juice, jelly, raisins, and wine, grapes surpass all other fruit crops in value. Therefore, successful efforts to improve grapevines are likely to have a major impact on commercial viticulture.
Current methods for improving grapevines are time-consuming and labor intensive. For example, genetic improvement in grapes through conventional breeding is severely limited by a number of factors such as long pre-bearing age and varying ploidy levels. Cultivated grapes are also highly heterozygous and do not generally breed true from seeds. Moreover, grape breeding programs are expensive, long-term projects. Although plant biotechnology is an attractive alternative for genetic improvement in grapes (Kuksova et al., Plant Cell Tiss. Org. Cult. 49:17-27, 1997), in vitro genetic manipulation can be addressed only if there is an effective regeneration system. Accordingly, methods that reduce any of these problems would represent a significant advancement in the art.
We have discovered methods for growing perennial grape embryogenic cultures and for growing large quantities of somatic grape embryos from such perennial embryogenic cultures in a relatively short period using a liquid suspension culture. Several advantages are provided by the present methods. These approaches, for example, facilitate an extraordinarily high frequency of somatic embryo formation and plant regeneration. Such frequencies have not been previously reported for grapevine regeneration of any known cultivar, and render the method useful for large-scale production of clonal planting stock of grape plants. In addition, the methods produce embryos free of such common abnormalities as fusion and fasciations of somatic embryos. The methods of the invention also result in enhanced embryogenic culture initiation frequency, allowing for the production of highly embryogenic cultures that can then be successfully carried through the subsequent stages of the regeneration process to the whole plant level. Because of these advantages, the methods of the invention are especially useful in the application of biotechnology for the genetic improvement of this crop.
Accordingly, in a first aspect, the invention features a method of producing a mature somatic embryo. The method includes the steps of: (a) providing a liquid culture that includes an embryogenic cell; (b) recovering embryogenic cell from the culture; (c) transferring the embryogenic cell to a second culture; and (d) growing a mature somatic grape embryo from the embryogenic cell.
Preferably, the embryogenic cell is obtained from anthers, ovaries, ovules, floral tissue, vegetative tissue, tendrils, leaves, roots, nucellar tissue, stems, seeds, protoplasts, pericycle, apical meristem tissue, embryogenic tissue, somatic embryos, or zygotic embryos. In still other preferred embodiments, the liquid culture medium of step (a) includes a plant growth regulator (e.g., an auxin, NOA (naphthoxyacetic acid), indole-3-acetic acid (IAA), dicamba, picloram, naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), a cytokinin, benzyladenine (BA), thidiazuron, zeatin, abscisic acid (ABA), or gibberellic acid (GA). In other preferred embodiments, the initiation culture medium includes at least 0.01 mg/L of a cytokinin; at least 0.1 mg/L of a carbohydrate; and at least 0.1 mg/L of a nitrogenous compound. Exemplary carbohydrates include sucrose, glucose, maltose, and glycerol. Exemplary nitrogenous compounds include potassium nitrate, calcium nitrate, anunonium nitrate, and ammonium sulfate. If desired, the embryogenic cell or the somatic embryo can be transformed with DNA (e.g., DNA encoding a gene which is capable of conferring disease resistance). The embryogenic cell can be recovered from the first culture by filtration, sedimentation, or selection. In addition, the first culture can be sieved to synchronize the formation of differentiated somatic grape embryos.
Preferably, the embryogenic cell of step (a) is obtained from a source selected from the group consisting of: an embryogenic cell produced according to steps (a) and (b) of the first aspect; a somatic grape embryo produced according to steps (a) to (d) of the first aspect; a mature somatic embryo produced according to method of the first aspect; a plantlet according to the fifth aspect; and anthers, ovaries, ovules, floral tissue, vegetative tissue, tendrils, leaves, roots, nucellar tissue, stems, seeds, protoplasts, pericycle, apical meristem tissue, embryogenic tissue, somatic embryos, or zygotic embryos from a plant produced from a mature somatic embryo produced according to the method of the first aspect.
In preferred embodiments, the method further includes transferring the somatic grape embryo to a germination medium to grow a grape plantlet.
In other preferred embodiments, the embryogenic cell, if desired, is cultured on a medium including a plant growth regulator.
The second culture can be a liquid culture or a solid culture. A preferred medium for liquid culture includes B-5 major salts and MS minor salts.
In a second aspect, the invention features a method of producing a somatic grape embryo. The method includes the general steps of: (a) providing a liquid culture comprising an embryogenic cell; (b) selecting and the embryogenic cell from the culture; (c) transferring the embryogenic cell to a second culture; and (d) growing a somatic grape embryo from the embryogenic cell.
In a third aspect, the invention features a method of producing a somatic grape embryo. The method includes the general steps of: (a) providing a liquid culture that includes an embryogenic cell and medium consisting of B-5 major salts and MS minor salts; (b) recovering the embryogenic cell from the culture; (c) transferring the embryogenic cell to a second culture; and (d) growing a somatic grape embryo from the embryogenic cell.
In a fourth aspect, the invention features a mature somatic grape embryo produced according to the method of the first aspect.
In a fifth aspect, the invention features a plantlet germinated from the mature somatic grape embryo of fourth aspect.
In a sixth aspect, the invention features a method for long-term storage of a mature somatic grape embryo. The method includes the general step of drying the embryo and storing the embryo at a temperature less than 8xc2x0 C.
Preferably, the mature somatic grape embryo is produced by the method of the first aspect.
In a seventh aspect, the invention features a method for direct seeding of a somatic grape embryo. The method includes the general step of placing said somatic grape embryo in potting medium comprising sand and potting mixture.
In an eighth aspect, the invention features a method of producing a somatic grape embryo using a liquid culture medium from a perennial grape embryogenic culture. The method includes the steps of: (a) culturing a perennial grape embryogenic culture in a first liquid culture medium to grow a cellular suspension culture, the first liquid culture medium including a plant growth regulator; (b) recovering an embryogenic cell or embryogenic cell mass from the cellular suspension culture; and (c) culturing the embryogenic cell or embryogenic cell mass from the cellular suspension culture of step (b) in a second liquid culture medium to produce a somatic grape embryo.
In preferred embodiments, the method further includes transferring the somatic grape embryo to a germination medium to grow a grape plantlet.
In other preferred embodiments of the eighth aspect, the plant growth regulator is an auxin, NOA, BA, zeatin, dicamba, picloram, NAA, IAA, 2,4-D, a cytokinin, thidiazuron, ABA, or GA. In yet other preferred embodiments, the liquid cell culture of step (a) is subcultured, and the recovery of the grape cell or grape cell cluster from the cellular suspension culture includes filtration, sedimentation, or selection. In other preferred embodiments, the second liquid culture medium fuirther includes a plant growth regulator (e.g., a cytokinin). The liquid cell culture of step (c) can be sieved to synchronize the formation of differentiated somatic grape embryos. If desired, the somatic grape embryo or embryogenic cell of step (a) can each be transformed with DNA. The embryogenic cell or embryogenic cell mass recovered in step (b) may also be transformed with DNA. Similarly, the recovered somatic embryo may transformed with DNA.
In a ninth aspect, the invention features a method of producing a somatic grape embryo using a solid culture medium from a perennial grape embryogenic culture. The method includes the steps of: (a) culturing a embryogenic cell in a first liquid culture medium to grow a cellular suspension culture, the first liquid culture medium including a plant growth regulator; (b) recovering an embryogenic cell or embryogenic cell mass from the cellular suspension culture; and (c) culturing the embryogenic cell or embryogenic cell mass from the cellular suspension culture of step (b) in a solid culture medium to produce a somatic grape embryo.
In preferred embodiments of the ninth aspect, the solid culture medium further includes a plant growth regulator (e.g., a cytokinin, ABA, or GA). In other preferred embodiments, the recovered embryogenic cell or embryogenic cell mass are sieved to synchronize the formation of mature somatic grape embryos.
In a tenth aspect, the invention features a method of selecting for a somatic grape embryo having resistance to a plant pathogen using a liquid culture medium. The method including the steps of: (a) culturing a somatic grape embryo in a first liquid culture medium to grow a cellular suspension culture, the first liquid culture medium including a plant growth regulator and a filtrate from a plant pathogen culture; (b) recovering an embryogenic cell or embryogenic cell mass from the cellular suspension culture; and (c) culturing the embryogenic cell or embryogenic cell mass in a second liquid culture medium to produce a somatic grape embryo.
In an eleventh aspect, the invention features a method for producing a grape plant having resistance to a plant pathogen. The method includes the general steps of: (a) culturing a somatic grape embryo in a first liquid culture medium to grow a cellular suspension culture, the first liquid culture medium including a plant growth regulator and a filtrate from a plant pathogen culture; (b) recovering an embryogenic cell or embryogenic cell mass from the cellular suspension culture; (c) culturing the grape cell or grape cell cluster in a second liquid culture medium to produce a grape somatic embryo; (d) recovering the somatic grape embryo, wherein the somatic grape embryo has resistance to the plant pathogen; and (e) growing a plant from the somatic grape embryo.
In preferred embodiments of the tenth or eleventh aspect, the method further includes transferring the somatic grape embryo to a germination medium to grow a grape plantlet. In other preferred embodiments, the pathogen filtrate is obtained from a virus, nematode, insect, fungus (e.g., Elsinoxc3xa ampelina), or bacterium. In other preferred embodiments, the plant growth regulator of step (a) is an auxin. In yet other preferred embodiments, the second liquid culture medium further includes a plant growth regulator.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.